Figure 4
Strain-specific sequence motif for the binding site for STAT6 transcription factor in the PBS region of clade A, G, and related CRFs (02_AG, 01_AE and 22_01A1). (a) Sequences from nucleotide + 197 to + 232 used for the fEMSA. The G/CRFs-5′IRDye-STAT6 sequence from Subtype G and non 12_BF CRFs used as a probe, includes the reported STAT6 canonical motif. Its equivalent from subtype B was used as a mutated competitor. (b) The CRFs-5′IRDye-STAT6 probe (lane 1) was incubated in the absence (lane 1) or with 5 µg of nuclear extract from IL-4 stimulated Cos 7 cells (Lanes 2–7). In this gel-shift experiment, we noted the presence of a non-specific band even in the absence of nuclear extract (lanes 1–8). This suggests that compounds enhancing binding conditions in this experiment might bind to the designed oligonucleotide. More importantly, we observed a specific band for the DNA probe-protein complex (lane 3). The specificity of STAT6 protein and DNA probe binding was shown by the non-disruption of the protein-DNA complex in the presence of a 200-fold excess of the mutated competitor (lane 2) or non-specific oligonucleotide (poly(dI-dC), lane 4) and by the disruption of the DNA–protein complex in the presence of 100-fold excess of the cold probe (CRFs-STAT6, lane 5). Additionally, we observed a supershift of the DNA–protein complex with anti-STAT6 antibody (lane 7), and not with anti-STAT 3 (lane 6) or non-specific IgG (lane 8). Surprisingly, no shift was detected with anti-Stat3 and anti-IgG antibodies (lanes 6 and 8). This might suggest that in these specific conditions, the presence of these later antibodies may disrupt the protein–DNA interaction, resulting in loss of the Stat6-DNA complex. To improve the conciseness and clarity, the image was cropped and the bands of interest that were non-adjacent in the original gel (Supplementary information S1 Fig. 4), were juxtaposed with a clear separation.
